High-resolution structure of a papaya plant-defence barwin-like protein solved by in-house sulfur-SAD phasing

Huet, Joëlle; Mbosso, Emmanuel Jean Teinkela; Soror, Sameh H.; Meyer, Franck; Looze, Yvan; Wintjens, René; Wohlkonig, A.

doi:10.1107/s0907444913018015

Cited by 18 publications

(23 citation statements)

References 84 publications

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“…Because of the high sulfur content of Sizzled, we used a SAD method for phasing. A dataset with a total oscillation range of 360°was acquired because high-redundancy data may increase signal-to-noise level for reflections and thereby anomalous scattering signals (36,37). This dataset had a low R merge factor of 5.1% for all reflections, a relatively high data multiplicity of about 26, and a considerable mean anomalous difference of 3.5% (supplemental Table S1).…”

Section: Structure Determination By In-house Sulfur-sad Phasingmentioning

confidence: 99%

The crystal structure of full-length Sizzled from Xenopus laevis yields insights into Wnt-antagonistic function of secreted Frizzled-related proteins

Zhang

et al. 2017

Journal of Biological Chemistry

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The Wnt-signaling pathway is crucial to cell proliferation, differentiation, and migration. The secreted Frizzled-related proteins (sFRPs) represent the largest family of secreted Wnt inhibitors. However, their function in antagonizing Wnt signaling has remained somewhat controversial. Here, we report the crystal structure of Sizzled from , the first full-length structure of an sFRP. Tethered by an inter-domain disulfide bond and a linker, the N-terminal cysteine-rich domain (CRD) and the C-terminal netrin-like domain (NTR) of Sizzled are arranged in a tandem fashion, with the NTR domain occluding the groove of CRD for Wnt accessibility. A Dual-Luciferase assay demonstrated that removing the NTR domain and replacing the CRD groove residues His-116 and His-118 with aromatic residues may significantly enhance antagonistic function of Sizzled in inhibiting Wnt3A signaling. Sizzled is a monomer in solution, and Sizzled CRD exhibited different packing in the crystal, suggesting that sFRPs do not have a conserved CRD dimerization mode. Distinct from the canonical NTR domain, the Sizzled NTR adopts a novel α/β folding with two perpendicular helices facing the central mixed β-sheet. The subgroup of human sFRP1/2/5 and Sizzled should have a similar NTR domain that features a highly positively charged region, opposite the NTR-CRD interface, suggesting that the NTR domain in human sFRPs, at least sFRP1/2/5, is unlikely to bind to Wnt but is likely involved in biphasic Wnt signaling modulation. In summary, the Sizzled structure provides the first insights into how the CRD and the NTR domains relate to each other for modulating Wnt-antagonistic function of sFRPs.

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Section: Structure Determination By In-house Sulfur-sad Phasingmentioning

confidence: 99%

The crystal structure of full-length Sizzled from Xenopus laevis yields insights into Wnt-antagonistic function of secreted Frizzled-related proteins

Zhang

et al. 2017

Journal of Biological Chemistry

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“…The PR-4 proteins are classified into classes I and II based on whether they have an N-terminal Hevein domain or not ( Neuhaus et al, 1996 ). Wheatwin1 and wheatwin2 belong to class II of the PR-4 proteins and show antifungal activity by inhibiting fungal hyphal growth ( Caruso et al, 1996 , 2001 ; Huet et al, 2013 ). Furthermore, wheatwin1 is able to digest RNA from wheat and Fusarium culmorum ( Caporale et al, 2004 ).…”

Section: Introductionmentioning

confidence: 99%

“…In addition, a PR-4 in Capsicum chinense has been shown to exhibit deoxyribonuclease and ribonuclease in in vitro assays ( Guevara-Morato et al, 2010 ). AtHEL, a class I PR-4 protein of Arabidopsis , was found to interact with a fungal lectin having deoxyribonuclease and ribonuclease activities ( Bertini et al, 2012 ; Huet et al, 2013 ).…”

Section: Introductionmentioning

confidence: 99%

The Novel Gene VpPR4-1 from Vitis pseudoreticulata Increases Powdery Mildew Resistance in Transgenic Vitis vinifera L.

et al. 2016

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Pathogenesis-related proteins (PRs) can lead to increased resistance of the whole plant to pathogen attack. Here, we isolate and characterize a PR-4 protein (VpPR4-1) from a wild Chinese grape Vitis pseudoreticulata which shows greatly elevated transcription following powdery mildew infection. Its expression profiles under a number of abiotic stresses were also investigated. Powdery mildew, salicylic acid, and jasmonic acid methyl ester significantly increased the VpPR4-1 induction while NaCl and heat treatments just slightly induced VpPR4-1 expression. Abscisic acid and cold treatment slightly affected the expression level of VpPR4-1. The VpPR4-1 gene was overexpressed in 30 regenerated V. vinifera cv. Red Globe via Agrobacterium tumefaciens-mediated transformation and verified by the Western blot. The 26 transgenic grapevines exhibited higher expression levels of PR-4 protein content than wild-type vines and six of them were inoculated with powdery mildew which showed that the growth of powdery mildew was repressed. The powdery mildew-resistance of Red Globe transformed with VpPR4-1 was enhanced inoculated with powdery mildew. Moreover, other powdery mildew resistant genes were associated with feedback regulation since VpPR4-1 is in abundance. This study demonstrates that PR-4 protein in grapes plays a vital role in defense against powdery mildew invasion.

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“…A recently described case of S-SAD phasing was associated with an R anom /R p.i.m. ratio as low as 1.1 (Huet et al, 2013;Lakomek et al, 2009). However, in these studies the R anom values were determined with SCALA based on the definition (Evans & Murshudov, 2013).…”

Section: Phasing Statisticsmentioning

confidence: 99%

Against the odds?De novostructure determination of a pilin with two cysteine residues by sulfur SAD

Gorgel

Bøggild

Ulstrup

et al. 2015

Acta Cryst D Biol Crystallogr

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Exploiting the anomalous signal of the intrinsic S atoms to phase a protein structure is advantageous, as ideally only a single well diffracting native crystal is required. However, sulfur is a weak anomalous scatterer at the typical wavelengths used for X-ray diffraction experiments, and therefore sulfur SAD data sets need to be recorded with a high multiplicity. In this study, the structure of a small pilin protein was determined by sulfur SAD despite several obstacles such as a low anomalous signal (a theoretical Bijvoet ratio of 0.9% at a wavelength of 1.8 Å), radiation damage-induced reduction of the cysteines and a multiplicity of only 5.5. The anomalous signal was improved by merging three data sets from different volumes of a single crystal, yielding a multiplicity of 17.5, and a sodium ion was added to the substructure of anomalous scatterers. In general, all data sets were balanced around the threshold values for a successful phasing strategy. In addition, a collection of statistics on structures from the PDB that were solved by sulfur SAD are presented and compared with the data. Looking at the quality indicator R(anom)/R(p.i.m.), an inconsistency in the documentation of the anomalous R factor is noted and reported.

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High-resolution structure of a papaya plant-defence barwin-like protein solved by in-house sulfur-SAD phasing

Cited by 18 publications

References 84 publications

The crystal structure of full-length Sizzled from Xenopus laevis yields insights into Wnt-antagonistic function of secreted Frizzled-related proteins

The crystal structure of full-length Sizzled from Xenopus laevis yields insights into Wnt-antagonistic function of secreted Frizzled-related proteins

The Novel Gene VpPR4-1 from Vitis pseudoreticulata Increases Powdery Mildew Resistance in Transgenic Vitis vinifera L.

Against the odds?De novostructure determination of a pilin with two cysteine residues by sulfur SAD

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